The focus of Project 2 of this Program Project Grant is to pursue new approaches to cultivate the human caliciviruses, which are Group B biodefense pathogens that can cause explosive, incapacitating outbreaks of nonbacterial gastroenteritis. Our long-term goal is to exploit two new, fundamental discoveries made in recent basic studies of human caliciviruses to develop needed gene expression and cultivation systems. These discoveries include the findings that (1) a cell binding ligand for Norwalk virus is a carbohydrate histo-blood group antigen, and (2) the small basic structural protein VP2 (the ORF3 protein) is a regulator of capsid stability that also is likely involved in encapsidation of the viral genome. In Specific aim 1, an in vitro method for cultivation of Norwalk virus and other noroviruses will be developed. Studies in Specific aim 2 will determine if intestinal epithelial cells or primary cells from humans will express Norwalk virus genes or replicate Norwalk virus when exposed to viral RNA or molecularly engineered virus gene constructs. In Specific aim 3, gene expression and replication systems will be used to dissect the biologic significance of the VP2 minor structural protein, including its phosphorylation and role in virus assembly. These studies are designed to understand the molecular basis of restriction of virus gene expression and growth in cell systems. They will complement and facilitate development of new, rapid methods of virus detection to be pursued in Project 1, as well as enhance the biologic interpretation of new structural data to be obtained in Project 3. A goal is to help design methods to prevent or treat disease and provide a molecular basis for understanding differences in calicivirus tropism and virulence. Together, results from these studies will help determine correlates of protection from infection and disease, and permit virus viability to be measured after exposure to different inactivating conditions. Our previous experience and availability of unique reagents to pursue these goals will assure rapid progress in understanding the viral and cellular molecules critical for regulating caliciviral replication, stability and pathogenesis.